Difference between revisions of "Team:ETH Zurich/Experiments/Anti Cancer Toxin"

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<p>Once a tumor environment has been recognized and colonized by our bacteria, the production of an anti-cancer toxin, together with an MRI contrast agent will be triggered. The module that we engineered to achieve this purpose includes a synthetic promoter ("Sensor module link") that recognizes the presence of high lactate and hign bacterial density in the tumor environment and allows the transcription of the anti-cancer toxin, Azurin, and the MRI contrast agent, bacterioferritin, from the same operon. </p>
 
<p>Once a tumor environment has been recognized and colonized by our bacteria, the production of an anti-cancer toxin, together with an MRI contrast agent will be triggered. The module that we engineered to achieve this purpose includes a synthetic promoter ("Sensor module link") that recognizes the presence of high lactate and hign bacterial density in the tumor environment and allows the transcription of the anti-cancer toxin, Azurin, and the MRI contrast agent, bacterioferritin, from the same operon. </p>
  
−
     <figure class="fig-nonfloat" style="width:700px;">
+
 
−
         <img src="https://static.igem.org/mediawiki/2017/3/32/T--ETH_Zurich--WL_TS_figure1.png">
+
     <figure class="fig-nonfloat" style="width:800px;">
−
         <figcaption>Figure 1. Depictions of the three designs of the AND-gates we characterized. Design a) is based on the part <a href="http://parts.igem.org/Part:BBa_K1847007"> BBa_K1847007</a> while designs b) and c) differ in the spacing after O2 and the numbers of O1 and O2 respecitvely. In all cases, only in case both inducers, AHL and Lactate, are present, the DNA should be unloope which would lead to exposure of the <a href="http://parts.igem.org/Part:BBa_R0062">Plux</a> promoter such that the dimerized <a href=http:"http://parts.igem.org/Part:BBa_C0062">LuxR</a> can activate expression of downstream genes.</figcaption>
+
         <img src="https://static.igem.org/mediawiki/2017/c/c9/T--ETH_Zurich--Circuit_MRI_figure3.png">
 +
         <figcaption>Figure 1. Change in signal generated by expression of bacterioferritin. Once the <a href="https://2017.igem.org/Team:ETH_Zurich/Circuti/Fa_Tumor_Sensor">Tumor Sensing</a> has been activated, both the <a href="https://2017.igem.org/Team:ETH_Zurich/Circuti/Fc_Anti_Cancer_Toxin">Anti-Cancer Toxin</a> azurin and the MRI Contrast Agent bacterioferritin are produced. Azurin will accumulate inside of the bacteria until it is ready for release. Bacterioferritin will take up iron and produce a change in the T2 signal in MRI.</figcaption>
 
     </figure>
 
     </figure>
  
−
<p>For more details about the reasoning about and functioning of our synthetic AND-gate promoter, see the <a href="https://2017.igem.org/Team:ETH_Zurich/Circuit/fa_TumorSensor">circuit page</a>.</p>
+
<p>For more details about bacterioferritin and its role in our system, go to our description of the <a href="https://2017.igem.org/Team:ETH_Zurich/Circuit/Fb_MRI_Contrast_Agent">MRI Contrast Agent</a>.</p>
 
</section>
 
</section>
  
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<h1>Overview of the Experiments</h1>
 
<h1>Overview of the Experiments</h1>
  
−
<p>In order to achieve building and characterizing an AND-gate that would allow for faithful differentiation between healthy and tumor tissue, we ran a sequence of experiments:
+
<p>Thanks to <a href="https://2017.igem.org/Team:ETH_Zurich/HP/Gold_Integrated#rudin-interview">professor Markus Rudin</a> and his team from the Animal Imaging Center at the  Institute for Biomedical Engineering of ETH Zurich, we were able to perform experiments in a small-animal MRI scanner. To test the consequences of bacterioferritin overexpression in an MRI scanner, we transformed <i>E. coli</i> Nissle with a plasmid containing an AHL-inducible promoter (pLux) that controls the expression of both a green fluorescent protein (GFP) and bacterioferritin (Figure 2). </p>
−
<ul>
+
−
<li>We transformed E. Coli with plasmids containing only the quorum sensing system. We let these colonies grow to different densities and evaluated the colonies' response to this. </li>
+
−
<li>We evaluated the response of our AND-gate designs to varying amounts of Lactate and AHL.</li>
+
−
<li>Finally, we transformed E. Coli with plasmids containing the whole tumor sensor system and evaluated the colonies' bevhaviour over time under conditions corresponding to healthy and tumorous tissue.</li>
+
−
</ul>
+
−
</p>
+
−
</section>
+
  
−
<section>
+
<figure class="fig-nonfloat" style="width:800px;">
−
<h1>Initial System Design</h1>
+
        <img src="https://static.igem.org/mediawiki/2017/6/6e/T--ETH_Zurich--Exp_MRI_figure2.png">
 +
        <figcaption>Figure 2. AHL diffuses into the cell and  binds to LuxR. The AHL/LuxR complex activates pLux, which results in transcription of both GFP and bacterioferritin.</figcaption>
 +
</figure>
  
−
<p><b>OBJECTIVE:</b> Before we started any designing, cloning or experimentation on the tumor sensor module, we sat together with our modellers to find key parameters relevant for design and experimentation.</p>
+
<p>First, we determined the concentration of AHL needed for full induction of the system. To do this, we measured changes in fluorescence caused by different concentrations of AHL used for induction. Second, we performed an SDS-PAGE analysis to confirm that bacterioferritin was indeed expressed along with GFP after induction with the appropriate concentration of AHL. Finally, we grew the bacteria in an iron-supplemented medium to observe the consequences of bacterioferritin overexpression on the MRI signal.</p>
  
−
<p><b>RESULTS:</b>
+
<p>To read more about each of these experiments, click on the buttons below. For a detailed protocol describing each experiment, visit <a href="https://2017.igem.org/Team:ETH_Zurich/Protocols">Protocols.</a></p>
−
<ul>
+
−
<li>Based on previous work done on quorum sensing: how strongly should LuxR and LuxI be expressed? <br>
+
−
Quick answer: each 10 times stronger than the ones characterized <a href="https://2014.igem.org/Team:ETH_Zurich/expresults">here</a>.</li>
+
−
<li>Similarly, what expression levels of LldP/LldR should be achieved in order to get enough sensitivity to differentiate tumor and non-tumor tissue?<br>
+
−
Quick answer: ...</li>
+
−
<li>At what density of the colony under experimental conditions should the quorum sensing system be activated?<br>
+
−
Quick answer: at an OD of 0.005. This turned out to be problematic to assess experimentally.</li>
+
−
</ul<
+
−
</p>
+
 
</section>
 
</section>
  
−
<section>
 
−
<h1>Quorum Sensing End-Point Characterization</h1>
 
  
−
<p><b>OBJECTIVE:</b> Determine the population density at which the quorum system gets activated and provide the modellers with data to infer a<sub>LuxI</sub>, the production rate of LuxI.</p>
+
<div class="multi-summary">
 +
<details>
 +
<summary>Fluorescence Measurement to Obtain the AHL Dose-Response Curve</summary>
 +
<p><b>OBJECTIVE</b><br>
 +
Determine the dose-response curve and the concentration of AHL needed for full induction of the system are by measuring the fluorescence after induction with different concentrations of AHL.</p>
  
−
<p><b>PROCEDURE (LINK)</b>We transformed E. Coli with a regulator and an actuator plasmid, containing constitutive LuxR and Plux, sfGFP, mCherry and LuxI respectively.  
+
<p><b>PROCEDURE</b><br>
 +
Biological triplicates of E. coli Nissle transformed with AHL-inducible promoter that controls the expression of bacterioferritin and GFP (Figure 2) were transferred in a 96-well plate and induced with twelve different concentrations of AHL (from 0 to 10<sup>-2</sup> M). Fluorescence and absorbance were measured in a plate reader over a period of 4 hours. A detailed protocol is available in<a href="https://2017.igem.org/Team:ETH_Zurich/Protocols">Protocols.</a></p>
  
−
    <figure class="fig-nonfloat" style="width:400px;">
+
<p><b>RESULTS</b><br> Based on measurements of fluorescence over time, a time point t = 200 minutes was chosen as representative of the plateau region. The relationship of fluorescence at that time point and the concentration of AHL used for induction was plotted to obtain the AHL dose-response curve (Figure 3).</p>
−
        <img src="https://static.igem.org/mediawiki/2017/9/9a/T--ETH_Zurich--WL_TS_figure2.png">
+
−
        <figcaption>Figure 2. Depictions of the two transformed plasmids. One contains the regulator, LuxR. The other one Plux which responds to dimerized LuxR. LuxR dimerizes upon binding to AHL which synthesis is catalyzed by LuxI.</figcaption>
+
−
    </figure>
+
  
−
Subsequently, we let these colonies grow to different final population densities. This was achieved by varying glucose concentrations in a defined medium [1]. Population density was assessed by measuring absorbance at 600 nm wavelength. As a read-out of the level of activation served fluorescence emitted by sfGFP and mCherry. LINK TO PROTOCOL.  </p>
 
−
 
−
<p><b>RESULTS:</b>
 
 
<figure class="fig-nonfloat" style="width:500px;">
 
<figure class="fig-nonfloat" style="width:500px;">
−
         <img src="https://static.igem.org/mediawiki/2017/3/3b/T--ETH_Zurich--QS_Trigger_Analysis.png">
+
         <img src="https://static.igem.org/mediawiki/2017/a/a1/T--ETH_Zurich--AHL_Dose_Response.png">
−
         <figcaption>Figure 2. A) Fluorescense per A600 in response to population density. Colonies were grown over night in media with varying glucose concentrations that lead to different final population denisities. With increasing absorbances at 600 nm, increasing fluorescence levels are observed. B) Proof of concept that final population densities can be modulated with the amount of glucose in a defined medium. </figcaption>
+
         <figcaption>Figure 3. AHL Dose-Response Curve obtained by measuring fluorescence.</figcaption>
 
</figure>
 
</figure>
−
</p>
 
  
−
<p><b>CONCLUSION:</b>
+
<p><b>CONCLUSION</b><br>  
−
<ul>
+
After consultation with the modelling team, we decided to use 1E-4 M of AHL for full induction of the system in future experiments. The measurements should be made at t = 200 minutes after induction.</p>
−
<li>We can modulate the density a bacterial population reaches in defined medium by varying the amount of glucose</li>
+
</details>
−
<li>The quorum sensing system shows a response to increasing population densities.</li>
+
−
</ul>
+
−
</p>
+
  
−
</section>
 
  
−
<section>
+
<details>
−
<h1>AND-gate without Quorum Sensing</h1>
+
<summary>SDS-PAGE to Confirm AHL-Induced Expression of Bacterioferritin</summary>
 +
 
 +
<p><b>OBJECTIVE</b><br>
 +
The concentration of AHL needed for full induction of the system was calculated based on fluorescence measurements and the assumption that bacterioferritin is co-expressed alongside GFP upon activation of pLux (Figure 2). To confirm that bacterioferritin is indeed co-expressed, an SDS-PAGE analysis is performed.</p>
 +
 
 +
<p><b>PROCEDURE</b><br>
 +
Protein lysates were obtained from bacteria treated with different concentrations of AHL. To determine the concentrations of proteins, needed to prepare the samples for SDS-PAGE, Bradford protein assay was preformed prior to the SDS-PAGE analysis. A detailed protocol is available in <a href="https://2017.igem.org/Team:ETH_Zurich/Protocols">Protocols.</a></p>
 +
 
 +
<p><b>RESULTS</b><br>
 +
To determine protein concentrations in the lysates via Bradford protein assay, a standard curve was  first generated by using a protein standard, bovine serum albumin, and measuring absorbance of different dilutions of the standard. Second, absorbance of the unknown samples was measured and the results were fitted to the curve (Figure 4).
  
−
<p><b>OBJECTIVE:</b> Determine expression levels of GFP production under the control of the AND-gate with different inducer concentrations. In this experiment we wanted to assess wheter our designs would be capable to distinguish healthy and tumor tissue based on lactate and expected AHL concentrations.
 
 
<figure class="fig-nonfloat" style="width:500px;">
 
<figure class="fig-nonfloat" style="width:500px;">
−
         <img src="https://static.igem.org/mediawiki/2017/5/56/T--ETH_Zurich--WL_TS_ANDgate_wo_LuxI.png">
+
         <img src="https://static.igem.org/mediawiki/2017/e/e1/ABS_vs_BSA.png">
−
         <figcaption>Figure x. Schematic depiction of the two plasmids that were transformed for this experiment. Both lactate and AHL were manually provided in this experiment.</figcaption>
+
         <figcaption>Figure 4. Standard curve of net absorbance versus the concentration of the protein standard (BSA = bovine serum albumin) needed for determination of protein concentration in the Bradford protein assay. </figcaption>
 
</figure>
 
</figure>
−
</p>
 
  
−
<p><b>PROCEDURE (LINK)</b>Cultures were grown in microtiter plates under combinations of 8 different AHL and 8 different lactate concentrations and measured after 5.5 hours. LINK TO PROTOCOL</p>
+
<p>After determining the unknown concentrations, samples were prepared accordingly and subjected to SDS-PAGE analysis. Bands sized approximately 18.4 kDa (which corresponds to bacterioferritin) were visible in the samples treated with AHL. There were no bands in untreated samples or the negative control (Figure 5).</p>
  
−
<p><b>RESULTS:</b> The different conditions cleary have an impact on expression levels of sfGFP under control of the AND-gate promoter. All three designs show increasing activation with increasing inducer concentration, even if the second inducer is not present. The highest fold-change for all designs however, is observed if both inducers are present in high amounts.</p>
+
<figure class="fig-nonfloat" style="width:500px;">
−
 
+
         <img src="https://static.igem.org/mediawiki/2017/6/6e/T--ETH_Zurich--Exp_SDSPAGE.png">
−
<figure class="fig-nonfloat" style="width:800px;">
+
         <figcaption>Figure 5. SDS-PAGE analysis of bacterioferritin expression upon AHL induction. (Bfr = sample from bacterioferritin-overexpressing bacteria, NC = negative control)</figcaption>
−
         <img src="https://static.igem.org/mediawiki/2017/a/a1/T--ETH_Zurich--AHL_Dose_Response.png">
+
−
         <figcaption>Figure 3. AHL Dose-Response Curve obtained by measuring fluorescence.</figcaption>
+
 
</figure>
 
</figure>
−
 
−
<p><b>CONCLUSION:</b>
 
−
<ul>
 
−
<li>Leakiness of the synthetic promoter increases with increasing amounts of either inducer in the absence of the other.</li>
 
−
<li>Increasing AHL amounts have a greater influence on the leakiness in absence of lactate.</li>
 
−
<li>All three AND-gates exhibit highest inductions in presence of both inducers.</li>
 
−
<li>At lactate levels found in healthy tissue and low AHL concentrations, all designs are only weakly activated.</li>
 
−
<li>Design b performed best at distinguishing “healthy tissue lactate”, low AHL vs. “tumor tissue lactate”, high AHL. Design c on the other hand performed worst.</li>
 
−
</ul>
 
 
</p>
 
</p>
  
−
</section>
+
<p><b>CONCLUSION</b><br>
 +
As expected, bands corresponding to bacterioferritin were visible in the samples induced with AHL. With this, to co-expression of bacterioferritin and GFP in the test strain for MRI experiments is confirmed.</p>
  
−
<section>
+
</details>
−
<h1>AND-gate with Quorum Sensing</h1>
+
  
−
<p><b>OBJECTIVE:</b> Verify the findings of the AND-gate characterization without quorum sensing with strains of E. Coli that contain additionally to the AND-gate also LuxI, the enzyme that catalzyes AHL production.</p>
+
<details>
 +
<summary>Magnetic Resonance Imaging of Bacterioferritin-Expressing <i>E. coli</i> Nissle</summary>
  
−
<p><b>PROCEDURE (LINK)</b>Cultures were grown in microtiter plates in media with varying lactate concentrations. Density and fluorescence measurements were taken every 15 minutes to ensure a high enough time-resolution. LINK TO PROTOCOL
+
<p><b>OBJECTIVE</b><br>
 +
To visualize  the signal change in MRI caused by overexpression of bacterioferritin, bacteria are grown in iron supplemented medium and imaged in an MRI scanner. </p>
  
−
<figure class="fig-nonfloat" style="width:400px;">
+
<p><b>PROCEDURE</b><br>
−
         <img src="https://static.igem.org/mediawiki/2017/2/21/T--ETH_Zurich--ANDgate_w_LuxI.png">
+
Biological triplicates of <i>E. coli</i> Nissle transformed with the heme-deleted bacterioferritin (Figure 2) were grown in four different experimental conditions (with and without induction and with and without iron supplementation) and imaged in a 4.7 T small animal MRI scanner. A bacterioferritin-expressing <i>E. coli</i> Top 10 (T7lacO-<i>bfr</i>) was used to compare the effect of the heme-deleted bacterioferritin against the wild-type bacterioferritin. Additionally, a <i>bfr</i>-knockout <i>E. coli</i> K-12 from the Keio collection was tested. A detailed protocol is available in <a href="https://2017.igem.org/Team:ETH_Zurich/Protocols">Protocols.</a></p>
−
         <figcaption>Figure y. Schematic depiction of the two plasmids that were transformed for this experiment. Lactate is provided to the system in this experiment, AHL is synthesized by the cells themselves.</figcaption>
+
 
 +
<p><b>RESULTS</b><br>
 +
All bacteria grown in iron-supplemented medium showed a drop in the T2 signal intensity, independent of induction of bacterioferritin expression. However, once induced, our bacteria experienced an additional drop in the signal, as predicted. The results are depicted as changes in the T2 relaxation rate, therefore a larger drop represents an increase in the change from the basal level, determined by imaging the bacteria grown without induction and without iron-supplementation (Figure 6).</p>
 +
 
 +
<figure class="fig-nonfloat" style="width:500px;">
 +
         <img src="https://static.igem.org/mediawiki/2017/8/8b/T--ETH_Zurich--BFR_T2_Change.png">
 +
         <figcaption>Figure 6. Influence of bacterioferritin overexpression on the MRI signal. T7lacO-<i>bfr</i> is a wild-type bacterioferritin-overexpressing strain, while pLux-<i>bfr M52H</i> represents our <i>E. coli</i> Nissle transformed with heme-deleted bacterioferritin under the control of an AHL-responsive promoter. The results are depicted as changes in the T2 relaxation rate, therefore a larger drop represents an increase in the change from the basal level, determined by imaging the bacteria grown without induction and without iron-supplementation. </figcaption>
 
</figure>
 
</figure>
  
−
</p>
+
<p>All the bacteria were resuspended and imaged in PBS, after washing of the culture medium. To test if any unwashed iron could mask the signal, the T2 relaxation rate was compared in pure PBS versus PBS supplemented with iron. Moreover, a <i>bfr</i>-knockout was imaged to see how much the endogenous bacterioferritin contributes to the signal when the bacteria are grown in the presence of iron. The results showed that the free iron in the medium only slightly changes the signal and should not interfere with the measurements. On the other hand, the <i>bfr</i>-knockout strain showed the same behaviour as the wild-type bacteria, suggesting that other iron-storage systems present in the bacteria contribute to iron uptake significantly (Figure 7). The differences in absolute values of the signal changes might be explained by the fact that different strains of <i>E. coli</i> were imaged. T7lacO-<i>bfr</i> is Top 10, pLux-<i>bfr M52L</i> is Nissle, while the <i>bfr</i>-knockout is K-12.</p>
  
−
<p><b>RESULTS:</b> The data is very noisy and it’s hard to make general statements about this systems behaviour. Despite this, a clear trend is visible for GFP to be higher expressed under lactate concentrations similar to tumor tissue than under those resembling healthy tissue or no lactate at all. With increasing population densities this effect becomes less pronounced.</p>
+
<figure class="fig-nonfloat" style="width:500px;">
−
 
+
         <img src="https://static.igem.org/mediawiki/2017/b/b7/T--ETH_Zurich--BFR_T2_Relax_all.png">
−
<figure class="fig-nonfloat" style="width:800px;">
+
         <figcaption>Figure 7. Influence of bacterioferritin and iron on the MRI signal. T7lacO-<i>bfr</i> is a wild-type bacterioferritin-overexpressing strain, while pLux-<i>bfr M52H</i> represents our <i>E. coli</i> Nissle transformed with heme-deleted bacterioferritin under the control of an AHL-responsive promoter.</figcaption>
−
         <img src="https://static.igem.org/mediawiki/2017/1/1b/T--ETH_Zurich--AND_vsLuxI_B_norm.png">
+
−
         <figcaption>Figure z. Fluorescence normalized to population density vs. population density. Blue circles correspond to media lacking lactate, green to media containing 1 mM lactate, and red to 5 mM lactate. Circle styles correspond to three different biological replicates. It becomes apparent that with higher densities comes higher activation and that for lower population densities, lactate has a positive influence on GFP expression levels.</figcaption>
+
 
</figure>
 
</figure>
  
−
<p><b>CONCLUSION:</b>  
+
<p><b>CONCLUSION</b><br>A decrease in signal intensity was observed for all bacteria grown in iron supplemented medium, probably due to presence of inherent bacterial iron-storage proteins. However, an additional drop in the signal was observed in samples where bacterioferritin overexpression was induced, as predicted. This result proves the usability of bacterioferritin as an MRI contrast agent in vitro and confirms the potential to use it as an in vivo reporter of tumor sensing.</p>
−
<ul>
+
−
<li>Due to a lot of noise in the data, conclusions have to be drawn with caution</li>
+
−
<li>Under lactate concentration mimicking tumor tissue, GFP gets stronger expressed than under lactate levels associated with healthy tissue.</li>
+
−
<li>Fold-changes are around 4 for design b and 2 for design a which is considerably less than observed in figure 3. This might be due to a somewhat different experimental setup.</li>
+
−
</ul>
+
−
</p>
+
−
 
+
−
</section>
+
  
 +
</details>
 +
</div>
  
 
<section class="references">
 
<section class="references">
 
     <h1>References</h1>
 
     <h1>References</h1>
 
     <ol>
 
     <ol>
−
         <li id="bib1"><a href="#ref1">^ </a>Contois, D. E. "Kinetics of bacterial growth: relationship between population density and specific growth rate of continuous cultures." Microbiology 21.1 (1959): 40-50.</li>
+
         <li id="bib1"><a href="#ref1">^ </a>Forbes, Neil S. "Engineering the perfect (bacterial) cancer therapy." <i>Nature reviews. Cancer</i> 10.11 (2010): 785.</li>
 +
        <li id="bib2"><a href="#ref2">^ </a>Cronin, M., et al. "Bacterial vectors for imaging and cancer gene therapy: a review." <i>Cancer gene therapy</i> 19.11 (2012): 731.</li>
 +
        <li id="bib3"><a href="#ref3">^ </a>Gilad, Assaf A., and Mikhail G. Shapiro. "Molecular Imaging in Synthetic Biology, and Synthetic Biology in Molecular Imaging." <i>Molecular Imaging and Biology</i> 19.3 (2017): 373-378.</li>
 +
        <li id="bib4"><a href="#ref4">^ </a>Lyons, Scott K., P. Stephen Patrick, and Kevin M. Brindle. "Imaging mouse cancer models in vivo using reporter transgenes." <i>Cold Spring Harbor Protocols</i> 2013.8 (2013): pdb-top069864.</li>
 +
        <li id="bib5"><a href="#ref5">^ </a>Cohen, Batya et al. “Ferritin as an Endogenous MRI Reporter for Noninvasive Imaging of Gene Expression in C6 Glioma Tumors.” <i>Neoplasia (New York, N.Y.)</i> 7.2 (2005): 109–117. Print.</li>
 +
        <li id="bib6"><a href="#ref6">^ </a>Hill, Philip J., et al. "Magnetic resonance imaging of tumors colonized with bacterial ferritin-expressing Escherichia coli." <i>PLoS One</i> 6.10 (2011): e25409.</li>
 
     </ol>
 
     </ol>
 
</section>
 
</section>

Revision as of 12:56, 28 October 2017

Anti-Cancer Toxin

Introduction

Once a tumor environment has been recognized and colonized by our bacteria, the production of an anti-cancer toxin, together with an MRI contrast agent will be triggered. The module that we engineered to achieve this purpose includes a synthetic promoter ("Sensor module link") that recognizes the presence of high lactate and hign bacterial density in the tumor environment and allows the transcription of the anti-cancer toxin, Azurin, and the MRI contrast agent, bacterioferritin, from the same operon.

Figure 1. Change in signal generated by expression of bacterioferritin. Once the Tumor Sensing has been activated, both the Anti-Cancer Toxin azurin and the MRI Contrast Agent bacterioferritin are produced. Azurin will accumulate inside of the bacteria until it is ready for release. Bacterioferritin will take up iron and produce a change in the T2 signal in MRI.

For more details about bacterioferritin and its role in our system, go to our description of the MRI Contrast Agent.

Overview of the Experiments

Thanks to professor Markus Rudin and his team from the Animal Imaging Center at the Institute for Biomedical Engineering of ETH Zurich, we were able to perform experiments in a small-animal MRI scanner. To test the consequences of bacterioferritin overexpression in an MRI scanner, we transformed E. coli Nissle with a plasmid containing an AHL-inducible promoter (pLux) that controls the expression of both a green fluorescent protein (GFP) and bacterioferritin (Figure 2).

Figure 2. AHL diffuses into the cell and binds to LuxR. The AHL/LuxR complex activates pLux, which results in transcription of both GFP and bacterioferritin.

First, we determined the concentration of AHL needed for full induction of the system. To do this, we measured changes in fluorescence caused by different concentrations of AHL used for induction. Second, we performed an SDS-PAGE analysis to confirm that bacterioferritin was indeed expressed along with GFP after induction with the appropriate concentration of AHL. Finally, we grew the bacteria in an iron-supplemented medium to observe the consequences of bacterioferritin overexpression on the MRI signal.

To read more about each of these experiments, click on the buttons below. For a detailed protocol describing each experiment, visit Protocols.

Fluorescence Measurement to Obtain the AHL Dose-Response Curve

OBJECTIVE
Determine the dose-response curve and the concentration of AHL needed for full induction of the system are by measuring the fluorescence after induction with different concentrations of AHL.

PROCEDURE
Biological triplicates of E. coli Nissle transformed with AHL-inducible promoter that controls the expression of bacterioferritin and GFP (Figure 2) were transferred in a 96-well plate and induced with twelve different concentrations of AHL (from 0 to 10-2 M). Fluorescence and absorbance were measured in a plate reader over a period of 4 hours. A detailed protocol is available inProtocols.

RESULTS
Based on measurements of fluorescence over time, a time point t = 200 minutes was chosen as representative of the plateau region. The relationship of fluorescence at that time point and the concentration of AHL used for induction was plotted to obtain the AHL dose-response curve (Figure 3).

Figure 3. AHL Dose-Response Curve obtained by measuring fluorescence.

CONCLUSION
After consultation with the modelling team, we decided to use 1E-4 M of AHL for full induction of the system in future experiments. The measurements should be made at t = 200 minutes after induction.

SDS-PAGE to Confirm AHL-Induced Expression of Bacterioferritin

OBJECTIVE
The concentration of AHL needed for full induction of the system was calculated based on fluorescence measurements and the assumption that bacterioferritin is co-expressed alongside GFP upon activation of pLux (Figure 2). To confirm that bacterioferritin is indeed co-expressed, an SDS-PAGE analysis is performed.

PROCEDURE
Protein lysates were obtained from bacteria treated with different concentrations of AHL. To determine the concentrations of proteins, needed to prepare the samples for SDS-PAGE, Bradford protein assay was preformed prior to the SDS-PAGE analysis. A detailed protocol is available in Protocols.

RESULTS
To determine protein concentrations in the lysates via Bradford protein assay, a standard curve was first generated by using a protein standard, bovine serum albumin, and measuring absorbance of different dilutions of the standard. Second, absorbance of the unknown samples was measured and the results were fitted to the curve (Figure 4).

Figure 4. Standard curve of net absorbance versus the concentration of the protein standard (BSA = bovine serum albumin) needed for determination of protein concentration in the Bradford protein assay.

After determining the unknown concentrations, samples were prepared accordingly and subjected to SDS-PAGE analysis. Bands sized approximately 18.4 kDa (which corresponds to bacterioferritin) were visible in the samples treated with AHL. There were no bands in untreated samples or the negative control (Figure 5).

Figure 5. SDS-PAGE analysis of bacterioferritin expression upon AHL induction. (Bfr = sample from bacterioferritin-overexpressing bacteria, NC = negative control)

CONCLUSION
As expected, bands corresponding to bacterioferritin were visible in the samples induced with AHL. With this, to co-expression of bacterioferritin and GFP in the test strain for MRI experiments is confirmed.

Magnetic Resonance Imaging of Bacterioferritin-Expressing E. coli Nissle

OBJECTIVE
To visualize the signal change in MRI caused by overexpression of bacterioferritin, bacteria are grown in iron supplemented medium and imaged in an MRI scanner.

PROCEDURE
Biological triplicates of E. coli Nissle transformed with the heme-deleted bacterioferritin (Figure 2) were grown in four different experimental conditions (with and without induction and with and without iron supplementation) and imaged in a 4.7 T small animal MRI scanner. A bacterioferritin-expressing E. coli Top 10 (T7lacO-bfr) was used to compare the effect of the heme-deleted bacterioferritin against the wild-type bacterioferritin. Additionally, a bfr-knockout E. coli K-12 from the Keio collection was tested. A detailed protocol is available in Protocols.

RESULTS
All bacteria grown in iron-supplemented medium showed a drop in the T2 signal intensity, independent of induction of bacterioferritin expression. However, once induced, our bacteria experienced an additional drop in the signal, as predicted. The results are depicted as changes in the T2 relaxation rate, therefore a larger drop represents an increase in the change from the basal level, determined by imaging the bacteria grown without induction and without iron-supplementation (Figure 6).

Figure 6. Influence of bacterioferritin overexpression on the MRI signal. T7lacO-bfr is a wild-type bacterioferritin-overexpressing strain, while pLux-bfr M52H represents our E. coli Nissle transformed with heme-deleted bacterioferritin under the control of an AHL-responsive promoter. The results are depicted as changes in the T2 relaxation rate, therefore a larger drop represents an increase in the change from the basal level, determined by imaging the bacteria grown without induction and without iron-supplementation.

All the bacteria were resuspended and imaged in PBS, after washing of the culture medium. To test if any unwashed iron could mask the signal, the T2 relaxation rate was compared in pure PBS versus PBS supplemented with iron. Moreover, a bfr-knockout was imaged to see how much the endogenous bacterioferritin contributes to the signal when the bacteria are grown in the presence of iron. The results showed that the free iron in the medium only slightly changes the signal and should not interfere with the measurements. On the other hand, the bfr-knockout strain showed the same behaviour as the wild-type bacteria, suggesting that other iron-storage systems present in the bacteria contribute to iron uptake significantly (Figure 7). The differences in absolute values of the signal changes might be explained by the fact that different strains of E. coli were imaged. T7lacO-bfr is Top 10, pLux-bfr M52L is Nissle, while the bfr-knockout is K-12.

Figure 7. Influence of bacterioferritin and iron on the MRI signal. T7lacO-bfr is a wild-type bacterioferritin-overexpressing strain, while pLux-bfr M52H represents our E. coli Nissle transformed with heme-deleted bacterioferritin under the control of an AHL-responsive promoter.

CONCLUSION
A decrease in signal intensity was observed for all bacteria grown in iron supplemented medium, probably due to presence of inherent bacterial iron-storage proteins. However, an additional drop in the signal was observed in samples where bacterioferritin overexpression was induced, as predicted. This result proves the usability of bacterioferritin as an MRI contrast agent in vitro and confirms the potential to use it as an in vivo reporter of tumor sensing.

References

  1. ^ Forbes, Neil S. "Engineering the perfect (bacterial) cancer therapy." Nature reviews. Cancer 10.11 (2010): 785.
  2. ^ Cronin, M., et al. "Bacterial vectors for imaging and cancer gene therapy: a review." Cancer gene therapy 19.11 (2012): 731.
  3. ^ Gilad, Assaf A., and Mikhail G. Shapiro. "Molecular Imaging in Synthetic Biology, and Synthetic Biology in Molecular Imaging." Molecular Imaging and Biology 19.3 (2017): 373-378.
  4. ^ Lyons, Scott K., P. Stephen Patrick, and Kevin M. Brindle. "Imaging mouse cancer models in vivo using reporter transgenes." Cold Spring Harbor Protocols 2013.8 (2013): pdb-top069864.
  5. ^ Cohen, Batya et al. “Ferritin as an Endogenous MRI Reporter for Noninvasive Imaging of Gene Expression in C6 Glioma Tumors.” Neoplasia (New York, N.Y.) 7.2 (2005): 109–117. Print.
  6. ^ Hill, Philip J., et al. "Magnetic resonance imaging of tumors colonized with bacterial ferritin-expressing Escherichia coli." PLoS One 6.10 (2011): e25409.